应用皮肤特异性SPRRIB基因修饰小鼠探讨表皮屏障形成的信号机制

The epidermal barrier is indispensable to the maintenance of organisms. In our previous study, we used genetic techniques to investigate the role of Cdc42 in epidermal homeostasis and its mechanisms. We specifically disrupted Cdc42 in skin epidermal basal cells by creating Cdc42flox/flox-Keratin 14-Cre+ mice. The skin barrier function of Cdc42-null mice was markedly impaired. RNA profiling was performed on biopsies taken from normal and Cdc42-null skin. We found that Small proline protein 1B(SPRR1B), a member of the certified envelope proteins, was up-regulated in Cdc42-null epidermis. SPRR1B has been identified to up-regulated in patients with the skin disorder such as the loosening of hyperkeratosis, psoriasis and so on, which might suggest that this gene is important in formation of the epidermal barrier. However, the specific functions of SPRR1B in epidermal barrier formation is unknow. In this study, firstly, we will establish the epidermis-specific conditional SPRR1B gene knockout/over expression mice models to explore the functions and mechanism of SPRR1B in normal epidermis tissues and skin development; Secondly, we will establish the epidermal-specific conditional Cdc42-SPRR1B double genes knockout mice model to explore the relationship of Cdc42 and SPRR1B. Thirdly, we will get a clear picture of interaction between SPRR1B, Cdc42 and MAPK in the regulation of epidermal barrier function. This research could provide a theoretical basis for the formation mechanism of the epidermal barrier and for targeted therapy for skin disorders.

表皮屏障是皮肤发挥防御功能的结构基础。课题组前期应用表皮特异性Cdc42敲除(KO)小鼠发现：KO小鼠胚胎发育中表皮屏障功能破坏；转录谱测序显示表皮角质套膜蛋白小富脯氨酸蛋白1B(SPRR1B)基因显著上调。SPRR1B是角质套膜形成的重要分子，在松解性角化过度症、银屑病等多种皮肤屏障相关疾病中表达均上调。我们的研究提示SPRR1B可能在皮肤发育及表皮屏障形成中发挥作用，但具体信号机制不清。为探讨SPRR1B在表皮屏障及皮肤发育的信号机制，本研究拟用Cre/loxP系统，构建表皮特异性SPRR1B基因条件性敲除/过表达模型，探究其在表皮屏障形成过程中的作用；并进一步结合Cdc42突变小鼠，探讨SPRR1B与Cdc42联合缺陷在表皮屏障形成的机制；继而聚焦SPRR1B阐明Cdc42,MAPK信号在表皮屏障发生的信号调控网络，为表皮屏障形成机制及靶向治疗提供理论依据。

表皮屏障是皮肤发挥防御功能的结构基础。课题组应用表皮特异性Cdc42敲除(KO)小鼠发现：KO小鼠胚胎发育过程表皮屏障功能破坏。利用转录谱测序显示：表皮角质套膜蛋白小富脯氨酸蛋白1B(SPRR1B)基因显著上调。SPRR1B是角质套膜形成的重要分子，在松解性角化过度症、银屑病等多种皮肤屏障相关疾病中SPRR1B基因表达均上调，提示：SPRR1B可能在表皮屏障破坏引起皮肤疾病过程中起重要调控，但SPRR1B在皮肤发生功能鲜为人知。本课题在上一个课题的基础上，进一步探讨Cdc42和SPRR1B在表皮屏障发生中的作用和相互之间的关系，并以此为基础探索皮肤屏障形成中可能的信号调控网络。首先，发现SPRR家族蛋白在表皮的发育中受到精确的调控，呈现时空动态变化。其次，发现SPRR1B过表达一方面可以引起表皮细胞的过度增殖，另一方面引起细胞紧密连接蛋白ZO-1、粘附连接蛋白E-钙粘蛋白和桥粒连接蛋白桥粒斑蛋白的表达下降。再次，研究发现SPRR1B或SPRR2D过表达是Cdc42敲除导致细胞间连接和皮肤屏障功能障碍的部分原因。最后，发现Cdc42通过MAPK-AP-1信号通路调节SPRR1的表达。综上所述，本研究揭示了Cdc42-p38/MAPK-AP-1通路影响表皮角质套膜结构蛋白SPRR家族异位表达，是调节表皮屏障形成和维持的重要信号调控机制。